Parkinson's disease (PD) is the most prevalent human neurodegenerative movement disorder and is characterized by a selective and progressive loss of the dopaminergic neurons. Mutations in the genes parkin and PTEN-induced putative kinase 1 (PINK1) result in autosomal recessive forms of PD. It has been suggested that parkin and Pink1 function in the same pathway in Drosophila, with Pink1 acting upstream of parkin. Previous work in our laboratory has shown the ability of parkin to rescue an alpha-synuclein-induced PD-like phenotype in Drosophila. To investigate the ability of Pink1 to protect against alpha-synuclein-induced toxicity, we have performed longevity, mobility, and histological studies to determine whether Drosophila Pink1 can rescue the alpha-synuclein phenotypes. We have found that overexpression of Pink1 results in the rescue of the alpha-synuclein-induced phenotype of premature loss of climbing ability, suppression of degeneration of the ommatidial array, and the suppression of alpha-synuclein-induced developmental defects in the Drosophila eye. These results mark the first demonstration of Pink1 counteracting PD phenotypes in a protein toxicity animal model, and they show that Pink1 is able to impart protection against potentially harmful proteins such as alpha-synuclein that would otherwise result in cellular stress.

ABSTRACT. Overexpression of the gene coding for α-synuclein has been shown to be an inherited cause of Parkinson disease. Our laboratory has previously co-expressed the parkin and Pink1 genes to rescue α-synuclein-induced phenotypes within a Drosophila model. To further investigate the effect of Pink1 in this model, we performed longevity and behavioral studies using several drivers to express the α-synuclein and Pink1 genes. Our findings showed that overexpression of Pink1 and overexpression of Pink1 with α-synuclein resulted in an increased lifespan when driven with the TH-Gal4 transgene. This increase in longevity was accompanied by an increased healthspan, as measured by mobility over time, suggesting that this is an example of improved functional aging. Our results indicate that, in the dopaminergic cells targeted by TH-Gal4, increased expression of α-synuclein and Pink1 together have a synergistic effect, allowing for enhanced protection and increased survival of the organism.

Pink1 has been linked to both autosomal recessive and sporadic forms of Parkinson disease. The Pink1 protein is thought to be involved in mitochondrial protection by interacting with parkin to prevent oxidative damage, maintain mitochondrial integrity and regulate mitophagy. Pink1 and parkin have been linked to components of the insulin receptor (INR) pathway, including PTEN, Akt and Foxo, but their effects in the INR pathway have been largely overlooked. To further investigate the roles of Pink1/parkin, we have performed co-expression studies to determine the effects Pink1 and parkin on the Foxo-induced phenotype of developmental defects in the Drosophila eye. We examined directed expression of Pink1, parkin, Pink1 or parkin mutants, and Pink1 or parkin interfering RNAs (RNAi) with the overexpression of Foxo in the developing eye of Drosophila. Our findings show that reduction of Pink1 suppresses the effects of Foxo overexpression, where co-overexpression with Pink1 or parkin increases the severity of the phenotype. This suggests that Pink1 and parkin are able to increase the proapoptotic effects of Foxo. Contrary to the view that Pink1 and parkin act exclusively as protective proteins in the cell, it is likely that the Pink1/parkin pathway is involved in aspects of cell fate decisions other than degrading toxic proteins and maintaining mitochondrial integrity.

Parkinson disease pathology often includes the presence of ubiquitin-positive, α-synuclein-enriched inclusions in the remaining neurons. Pink1 (also identified as PARK6) encodes a serinethreonine kinase involved in mitochondrial protection that works with parkin to ubiquitinate various proteins, promoting mitophagy. The parkin protein works to tag cystolic proteins for degradation, and previous work in our laboratory has shown the ability of parkin to rescue a Gal4induced phenotype. To further investigate the role of Pink1 in protection against toxic proteins, we have performed expression studies to determine the effects of increases and decreases in Pink1 on the Gal4-induced phenotype consisting of developmental defects in the Drosophila eye. Our results show that Pink1 is able to rescue the Gal4-induced phenotype, highlighting a protective role for Pink1 against toxic proteins. When expressing low levels of Gal4, reductions in Pink1 or parkin are not able to induce a phenotype. This suggests that Pink1 or parkin may counter Gal4 effects despite reductions, or that the effects of low level Gal4 may be alleviated by an alternative mechanism. Moreover, the Pink1 mechanism of action during differing types of cell stress, including degradation of toxic proteins, warrants further investigation.